The long-term objective of this project is to further elucidate the roles of mechanical forces for chromosomes and chromosomal reactions. Chromosome morphogenesis and dynamics will be examined from this perspective, comparatively for E.coli versus the prophase-to-prometaphase transition of eukaryotic chromosomes. Approaches will focus on high resolution 3D analysis, over time in the cell cycle, in wild-type and mutant situations, in living cells where ever possible. For E.coli, specific issues to be addressed will include the underlying physical and molecular basis for development of the nucleoid's helical ellipsoidal shape/density, the significance of this shape for dynamic chromatin movements, and the possibility that of cell division-related licensing of replication initiation. For eukaryotic chromosomes, the possibility of a meiosis-like intermediate will be explored. In complementary studies, our beads and bottlenecks magnetic micropiston will be used to probe the effects of compression and confinement on nucleoids/chromatin and DNA, including effects of real-time changes in buffer and molecular conditions. The roles of mechanical force for RecA- mediated homology recognition and strand exchange will be examined by parallel single molecule studies. Individual steps will be isolated and studied. The notion that reactions mediated by protein phosphatase PP2A are governed in important ways by the elasticity of its HEAT repeat scaffolding subunit will be further explored. Approaches to be used will include in vitro AFM analysis, in silico molecular dynamics, in vivo studies analysis of the PP2A-mediated response to spindle tension during the second division of meiosis, with budding yeast as an experimental system.